Project Summary Newborn screening for Cystinosis, Wilson disease (WD) and primary immunodeficiencies (PIDD) is critical, because these disorders are devastating and fatal despite the existence of validated treatments. Unfortunately, most patients with these disorders are diagnosed after developing significant complications because of a lack of cost-effective screening methods. Patients treated before the onset of these negative sequalae have significantly improved outcomes. Many congenital disorders are caused by mutations that result in absent or diminished levels of proteins; thus, protein biomarkers have enormous potential in the diagnosis/screening of congenital disorders. As proof-of-concept, we have demonstrated that liquid chromatography combined with tandem mass spectrometry analysis of signature peptides can identify patients lacking specific protein markers of three life-threatening PIDD, cystinosis and WD. We believe that novel targeted proteomic analyses can be utilized as rapid, multiplexed, inexpensive approaches to screen for a broad variety of treatable congenital disorders. Our ultimate goal is to develop targeted proteomic methods into high-throughput screens that fit readily into established newborn screening workflows and guidelines. Our methods involve direct quantification of extremely low abundance proteins using immuno-affinity-based enrichment combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) applied to dried blood spots on newborn screening cards. The objective of the current application is to drastically reduce the timer-per-sample for this proteomic analysis so that it can be used for population-based screening. We have previously demonstrated the ability to multiplex targets for the screening of numerous congenital disorders in a single patient in a single run. Our central hypothesis is that a novel peptide tagging procedure can be applied to the proteomic workflow for the multiplexing for multiple patients so that it is appropriate for NBS. Our specific aims are: (1): Development of novel peptide tags and specific elution of peptides from monoclonal antibodies in order to reduce the NBS run-time per sample. We will develop the use of a novel set of peptide tags so that several newborns can be analyzed in the same mass spectrometry run in order to multiplex patient samples and bring method runtimes to a target value of < 2 min per newborn. A second component of this proteomic method optimization is the use of specific elution of target peptides from monoclonal antibodies after enrichment to further reduce sample complexity and runtime. (2): Performance testing, optimization and validation in normal control samples. The main metric that we will examine is the within-newborn reproducibility. The second metric is to gauge the level of interference from non-target peptides across a panel of newborns. (3): Multi-dimensional multiplex validation in blinded sets of various patient dried blood spot samples. (4): Pilot study to estimate the rate of false positives. We will test the fully optimized multiplex proteomic method on dried blood spots from 10,000 random newborns obtained from the Washington State NBS laboratory.